Monocyte derived cells with immunostimulating properties, their preparation and uses

ABSTRACT

The invention relates to stimulated monocyte derived cells presenting the following characteristics: 1) increased release, with respect to normal monocyte derived cells, of for instance PDGF (platelet derived growth factor), and increased presence, on their membranes, with respect to normal monocyte derived cells, of for instance CD1α, and/or: 2) presence in their nucleus of at least one exogenous nucleic acid which has been integrated in the absence of the monocyte derived cell division. These stimulated monocyte derived cells can be the active substance of pharmaceutical compositions.

The invention relates to stimulated monocyte derived cells, processesfor their preparation, and pharmaceutical compositions containing thesame.

It has long been established that macrophages have a primary role inwound and tissue repair (see for example Wong and Wahl, Inflammation andrepair, in Handbook of Exp Pharmacol, 95:509-548, 1990). They areinducers and regulators of healing; they favor angiogenesis and recruitcells which will complete wound repair. After a tissue has been injured(burn, ulcers, wounds, trauma, mucosal damage, infarcts or evenreconstructive surgery), macrophages called locally clean the wound byelimination of the necrotic debris formed by dead cells (by phagocytosisand proteolysis). At the same time macrophages, activated locally bythis phagocytosis, actively release growth factors, monokines andchemokines. These autologous factors stimulate surrounding cells tomultiply and to migrate towards the wound and to replace the dead cells.

Macrophages play a major role in the antitumoral response, and they areable to be activated by immunological activators against cancer cells(Adams D. and Hamilton T.: “Activation of macrophages for tumor cellkill: effector mechanism and regulation”; in Heppner & Fulton (eds),Macrophages and cancer. CRC Press, 1988, p. 27; Fidler M. Macrophagesand metastases. A biological approach to cancer therapy. Cancer Res. 45:4714, 1985).

Furthermore, macrophages, or other cells derived from monocytes or fromtheir precursors, with their strong capacity for endocytosis, digestion,and surface antigen presentation, are capable of inducing a specificimmune response. In this way, they represent good candidates for thepreparation of vaccines, and more specifically cellular autologousvaccines.

Monocytes derived cells (MDCs) are immune cells such as obtained byculture of blood mononuclear cells in non adherent gas permeable plasticor Teflon bags for 5 to 10 days at 37° C. in O₂/CO₂ atmosphere. Theirculture medium (RPMI. IMDM, AIM5 (Gibco) or X-VIVO (Biowhittaker))contains eventually cytokines or ligands as defined in patents noPCT/EP93101232, no WO94/26875 or EP 97/02703 or in the articlesmentioned below:

“Autologous lymphocytes prevent the death of monocytes in culture andpromote, as do GM-CSF, IL-3 and M-CSF, their differentiation intomacrophages”. (Lopez M., Martinache Ch., Canepa S., Chokri M., ScottoF., Bartholeyns J.; J. of Immunological Methods, 159: 29-38, 1993);

“Immune therapy with macrophages: Present status and criticalrequirements for implementation” (Bartholeyns J., Romet-Lemonne J-L.,Chokri M., Lopez M.; Immunobiol., 195: 550-562, 1996);

“In vitro generation of CD83⁺ human blood dendritic cells for activetumor immunotherapy” (Thurnher M., Papesh C., Ramoner R., Gastlt G. andal.; Experimental Hematology, 25:232-237, 1997);

“Dendritic cells as adjuvants for immune-mediated resistance to tumors”(Schuler G. and Steinman R. M.; J. Exp. Med., 186:1183-1187, 1997).

All these patent applications and articles are included herein forreferences.

They can be activated by IFN-γ at the end of culture to obtain inparticular cytotoxic macrophages. They can be entrifuged to beconcentrated and purified before resuspension in isotonic solution.

Monocyte derived cells (MDCs) can either be killer macrophages,phagocytozing cells, growth factors and cytokines releasing cells, ordendritic cells according to their conditions of differentiation.Dendritic cells can for example be obtained as described in “In vitrogeneration of CD83+ human blood dendritic cells for active tumorimmunotherapy” (Thurnher M., Papesh C., Ramoner R., Gastlt G. and al.;Experimental Hematology, 25:232-237, 1997) and “Dendritic cells asadjuvants for immune-mediated resistance to tumors” (Schuler G. andSteinman R. M.; J. Exp. Med., 186:1183-1187, 1997), and EP 97/092703.

Mature dendritic cells are very potent antigen presenting cells toinitiate an immune response. The dendritic cells can be characterized bythe induction of T cell proliferation and by their phenotype (presenceof CD80, CD86, CD83, MHC-I, MHC-II on their membranes).

The dendritic cells play an important role, but are difficult to obtainin large quantities, necessary for therapeutic purpose in particularbecause of the required presence of multiple cytokines. Moreover, thedendritic cells obtained according to standard procedures are notstimulated and therefore do not present direct anti-tumoral or tissuerepair properties.

One of the aim of the invention, is to provide stimulated monocytederived cells having enhanced biological activities as described above,when compared to monocyte derived cells described until now.

Another aim of the invention is to provide processes for the preparationof said stimulated monocyte derived cells.

Another aim of the invention is to provide new pharmaceuticalcompositions containing said stimulated monocyte derived cells.

Another aim of the invention is to provide new methods for the treatmentof tissue injuries.

Another aim of the invention is to provide new methods for the treatmentor vaccination against tumors or infectious (bacterial or viral)diseases.

Another aim of the invention is to provide new methods for gene therapy.

The invention relates to stimulated monocyte derived cells presentingthe following characteristics:

1)—increased release, with respect to normal monocyte derived cells, ofat least one of the following polypeptides, proteins or compounds:

PDGF (platelet derived growth factor) IGF1 (insulin growth factor) MDGF(macrophage derived growth factor) bFGF (basic fibroblast growth factor)GM-CSF (granulocyte macrophage - colony stimulating factor) heat shockor stress proteins, chemokines and monokines such as IL12 and IFNγ

enzymes or enzyme inhibitors,

complement components,

transfer proteins,

peroxides, NO (nitrous oxide),

bioactive lipids,

hormones,

 and

increased presence, on their membranes, with respect to normal monocytederived cells, of at least one of the following activation markers:CD1α, CD11a, CD80, CD83, CD86, MHC class I and MHC class II molecules,adhesions, or accessory molecules for immunostimulation such as ICAM, orCD40,

 and/or

2) presence in their nucleus of at least one exogenous nucleic acidwhich has been integrated in the absence of the monocyte derived celldivision.

The expression normal monocyte derived cells correspond to monocytescultured in defined media or in the presence of cytokines which have notbeen specifically stressed and therefore which do not release increasedlevels of immunostimnulatory proteins or compounds and simultaneously donot express markedly increased levels of MHC and accessory molecules ontheir membranes.

NO is usually not released due to NO synthase suppression, but, in thecase of the invention, NO synthase is uninhibited which causes releaseof NO.

Monocytes derived cells can be obtained for instance from blood derivedmonocytes purified and cultured in the presence of GM-CSF and anothercytokine, such as IL-4 or IL-13.

The invention relates more particularly to stimulated monocyte derivedcells as described above, wherein the released polypeptides, proteinsand compounds are those listed on Table 1.

According to an advantageous embodiment, tie activation markers arepresent in an amount of at least about 1000 molecules/cells.

This can be measured by flow cytometry.

In a particular embodiment of the invention, the monocyte derived cellsas described above contain exogenous compounds in their cytoplasm suchas drugs, protein and growth factors of interest.

In another embodiment, the monocyte derived cells as described abovecontain in their cytoplasm exogenous DNA coding for a protein ofinterest.

It should be made clear that depending upon the nature of the physicalstress, either the DNA contained in the cytoplasm of said monocytederived cells remain in the cytoplasm after the physical stress, orthere is an uptake of said exogenous DNA by their nucleus which is madepossible by the physical stress.

The physically stimulated monocyte derived cells of the invention areparticularly suited for vaccination purpose since they express at thesame time the antigen introduced, increased membrane levels of MHCmolecules and accessory molecules to interact with lymphocytes and theyrelease increased amounts of TH1 type (e.a. IL-12) cytokines.

According to an advantageous embodiment, the stimulated monocyte derivedcells of the invention, present the following characteristics:

1) increased release, with respect to normal monocyte derived cells, ofat least one of the following polypeptides or proteins:

PDGF (platelet derived growth factor) IGF1 (insulin growth factor) MDGF(macrophage derived growth factor) bFGF (basic fibroblast growth factor)GM-CSF (granulocyte macrophage - colony stimulating factor) heat shockor stress proteins, such as HSP70, HSP90, GP96, chemokines and monokinessuch as IL12 and IFNγ

 and

increased presence, on their membranes, with respect to normal monocytederived cells, of at least one of the following activation markers:CD1α, CD11, CD80, CD83, CD86, MHC class I and MHC class II molecules,adhesions, or accessory molecules for immunostimulation such as ICAM, orCD40,

 and/or

2) presence in their nucleus of at least one exogenous nucleic acidwhich has been integrated in the absence of the monocyte derived celldivision.

According to an advantageous embodiment of the invention, saidactivation markers are present in an amount of at least about 1000molecules/cell.

This can be measured by flow cytometry.

Advantageous stimulated monocyte derived cells according to theinvention present at least one of the following characteristics:

increased release, with respect to normal monocyte derived cells, of atleast one of the following polypeptides, proteins or compounds:

PDGF

IGF1

MDGF

bFGF

GM-CSF

heat shock or stress proteins

chemokines and monokines such as IL12 and IFNγ

enzymes or enzyme inhibitors

complement components

transfer proteins

peroxides, NO (nitrous oxide),

bioactive lipids

hormones

 and

increased presence, on their membranes, with respect to normal monocytederived cells, of at least one of the following activation markers:CD1α, CD11a, CD80, CD83, CD86, MHC class I and MHC class II molecules,adhesions, or accessory molecules for immunostimulation such as ICAM, orCD40.

According to an advantageous embodiment of the invention, the abovepolypeptides, proteins or compounds are present in an amount higher thanabout 1 pg/cell/hr. and the above activation markers are present in therange of about 10³ to about 10⁵ molecules/cell.

This can be measured by flow cytometry.

Advantageous stimulated monocyte derived cells of the invention presentat least one of the following characteristics:

increased release, with respect to normal monocyte derived cells, of atleast one of the following polypeptides or proteins:

PDGF

IGF1

MDGF

bFGF

GM-CSF

heat shock or stress proteins

chemokines and monokines such as IL12 and IFNγ

 and

increased presence, on their membranes, with respect to normal monocytederived cells, of one of the following activation markers: CD1α, CD11a,CD80, CD83, CD86, MHC class I and MHC class II molecules, adhesions, oraccessory molecules for immunostimulation such as ICAM, or CD40.

According to an advantageous embodiment of the invention, the abovepolypeptides, proteins or compounds are present in an amount higher thanabout 1 pg/cell/hr, and the above activation markers are present in therange higher than about 10³ and particularly of about 10³ to about 10⁵molecules/cell.

This can be measured by flow cytometry.

The invention relates more particularly to stimulated monocyte derivedcells, which present the characteristic of having integrated at leastone exogenous nucleic acid in their nucleus in the absence of themonocyte derived cell division.

It is to be reminded that transfer of exogenous nucleic acids in cellnuclei by non viral techniques can be effectively achieved in rapidlydividing cells. In non dividing cells such as those derived frommonocytes, the exogenous nucleic acids are internalized in vacuoles orin the cytoplasm, but very low integration in endogenous nucleic acidsand expression of the coded peptide occur (<5%). The physicalstimulation of the invention allows migration of the exogenous nucleicacids internalized from the cytoplasm to the nucleus and thereforeenables increased expression of the transgene.

Advantageous stimulated monocyte derived cells according to theinvention, present the following characteristics:

increased release, with respect to normal monocyte derived cells, of atleast one of the following polypeptides, proteins or compounds:

PDGF

IGF1

MDGF

bFGF

GM/CSF

heat shock or stress proteins

chemokines and monokines such as IL12 and IFNγ

enzymes or enzyme inhibitors

complement components

transfer proteins

peroxides, NO (nitrous oxide),

bioactive lipids

hormones;

and increased presence, on their membranes, with respect to normalmonocyte derived cells, of at least one of the following activationmarkers: CD1α, CD11a, CD80, CD83, CD86, MHC class I and MHC class IImolecules, adhesions, or accessory molecules for immunostimulation suchas ICAM, and CD40,

and presence in their nucleus of at least one exogenous nucleic acidwhich has been integrated in the absence of the monocyte derived celldivision.

According to an advantageous embodiment of the invention, theabove-mentioned polypeptides, proteins or compounds are present in anamount higher than about 1 pg/cell;hr, and the above-mentionedactivation markers are present in the range of about 10³ to about 10⁵molecules/cell.

This can be measured by flow cytometry.

Advantageous stimulated monocyte derived cells of the invention presentthe following characteristics:

increased release with respect to normal monocyte derived cells of atleast one of the following polypeptides or proteins:

PDGF

IGF1

MDGF

bFGF

GM-CSF

heat shock or stress proteins

chemokines and monokines such as IL12 and IFNγ

and increased presence on their membranes with respect to normalmonocyte derived cells of at least one of the following activationmarkers: CD1α, CD11a, CD80, CD83, CD86, MHC class I and MHC class IImolecules, adhesions, or accessory molecules for immunostimulation suchas ICAM, and CD40,

and presence in their nucleus of at least one exogenous nucleic acidwhich has been integrated in the absence of the monocyte derived celldivision.

According to an advantageous embodiment of the invention, theabove-mentioned polypeptides, proteins or compounds are present in anamount higher than about 1 pg/cell/hr, and the above-mentionedactivation markers are present in the range of about 10³ to about 10⁵molecules/cell.

The amount of polypeptides, proteins or compounds can be measured byELISA method and the number of membrane activation markers can bemeasured by flow cytometry.

The invention also relates to a process for the preparation of monocytesderived cells comprising the step of stimulation of said monocytederived cells by physical means such as: thermal stress (heating at 40°C. to 50° C. for at least 30 minutes), pressure change (from about 1 barto about 0.05 bar, or from about 1 bar to about 10 bars), microwaves,electric shock (about 1 to about 10 s at about 250 mV), orelectropulsation.

Thermal stress or heat shock is applied as described in: “Differentialinduction of stress proteins and functional effects of heat shock inhuman phagocytes.” (Polla B. S., Stubbe H., Kantengwa S.,Maridonneau-Parini I., Jacquier-Sarlin M. R.—Inflammation, 19:363-378,1995) or in “Stress-inducible cellular responses” (Feige U., Morimoto R.I., Yahara I., Polla B. S.—BirkhäuserVerlag (Basel, Boston, Berlin),492p., 1996).

Microwaves are applied under the following conditions: (5 sec to 5 min)500 to 750 Watts, repeated 1 to 5 times.

Electropulsation (for instance 5 to 10 square electric pulses of 5millisec at 0.3 to 0.8 kV/cm) allows flux of ions and of nucleic acidsand/or protein transporters from the cytoplasm through the nucleuspores. This positive flux is stopped after the pulsation and theexogenous nucleic acid is integrated in nuclear DNA (“Specificelectropermeabilization of leucocytes in a blood sample and applicationto large volumes of cells”; S. Sixou and J. Teissié; Elsevier,Biochimica et Biophysica Acta. 1028:154-160, 1990).

Electric shock is applied as described in “Control by Pulse Parametersof Electric Field-Mediated Gene Transfer in Mammalian Cells” (HendrickW. et al., Biophysical Journal, Vol. 66:524-531, February 1994).

The process for the preparation of stimulated monocyte derived cells ofthe invention comprises the following steps:

preparation of monocyte derived cells according to the following method:

1) recovery of blood derived mononuclear cells directly from bloodapheresis or from blood bag collection, followed if necessary bycentrifugation, to eliminate a substantial part of red blood cellsgranulocytes and platelets, and collection of peripheral bloodleukocytes;

2) washing peripheral blood leukocytes obtained at the preceeding stepsfor instance by centrifugation (to remove 90% of platelets, red bloodcells and debris) to obtain mononuclear cells;

3) resuspension of the total mononuclear cells (monocytes+lymphocytes)obtained at the preceding step in culture medium (AIM-V, RPMI or IMDMtype) at 10⁶ to 2.10⁷ cells/ml, possibly completed by cytokines and/orautologous serum, and culture for 5 to 10 days at 37° C. under O₂/CO₂atmosphere in hydrophobic gas permeable bags, to obtain monocyte derivedcells and contaminating lymphocytes;

stimulation of said monocyte derived cells by physical means such as:thermal stress (heating at 40° C. to 50° C. for at least 30 minutes),pressure change (from about 1 bar to about 0.05 bar, or from about 1 barto about 10 bars), microwaves, electric shock (about 1 to about 10 s atabout 250 mV), or electropulsation for a time sufficient to induce theabove-mentioned characteristics.

Stimulation of said monocyte derived cells can also be achieved by meansof chemicals which cause maturation of said monocyte derived cells,resulting in increased stimulation of cells as described above.Advantageous chemicals are those which are responsible for theproduction by monocyte derived cells of IFN which endogenously activatesthe cells (stress signal). This IFN induction can be generated by doublestranded RNA (such as poly IC) (polyinosinic-polycytidylic acid) or bybacterial or mycobacterial extracts and particularly bacterial type DNAor corresponding natural or chemically modified oligonucleotides.

The invention also relates to a process for the preparation ofstimulated monocyte derived cells, comprising the following steps:

preparation of monocyte derived cells according to the following method:

1) recovery of blood derived mononuclear cells directly from bloodapheresis or from blood bag collection, followed if necessary bycentrifugation, to eliminate a substantial part of red blood cellsgranulocytes and platelets, and collection of peripheral bloodleukocytes;

2) washing peripheral blood leukocytes obtained at the preceding stepsfor instance by centrifugation (to remove 90% of platelets, red bloodcells and debris) to obtain mononuclear cells;

3) resuspension of the cells (monocytes+lymphocytes) obtained at thepreceeding step in culture medium (AIM V, RPMI or IMDM type) at 10⁶ to2.10⁷ cells/ml, possibly completed by cytokines and/or autologous serum,and culture for 5 to 10 days at 37° C. under O₂/CO₂ atmosphere inhydrophobic gas permeable bags, to obtain monocyte derived cells andcontaminating lymphocytes;

stimulation of said monocyte derived cells by addition of chemicalswhich induce endogenous IFN production such as double stranded RNA orbacterial or mycobacterial extracts and particularly bacterial type DNAor corresponding natural or chemically modified oligonucleotides.

It should be noted that the presence of contaminating lymphocytes withthe monocyte derived cells during culture and differentiation of themonocytes allows a better control of stimulation and cell recoverythrough paracrine cellular interactions.

The lymphocytes are segregated from the stimulated monocytes derivedcells at the end of the process.

The monocyte derived cells can be for instance prepared according to amethod such as described in patents PCT/EP93/01232, WO94/26875 or EP97/02703 or in the articles mentioned below:

“Autologous lymphocytes prevent the death of monocytes in culture andpromote, as do GM-CSF, IL-3 and M-CSF, their differentiation intomacrophages”. (Lopez M., Martinache Ch., Canepa S., Chokri M., ScottoF., Bartholeyns J.; J. of Immunological Methods, 159:29-38, 1993);

“Immune therapy with macrophages: Present status and criticalrequirements for implementation” (Bartholeyns J., Romet-Lemonne J-L.,Chokri M., Lopez M.; Immunobiol., 195:550-562, 1996);

“In vitro generation of CD83⁺ human blood dendritic cells for activetumor immunotherapy” (Thurnher M., Papesh C., Ramoner R., Gastlt G. andal.; Experimental Hematology, 25:232-237, 1997);

“Dendritic cells as adjuvants for immune-mediated resistance to tumors”(Schuler G. and Steinman R. M.; J. Exp. Med., 186:1183-1187, 1997).

The monocyte derived cells and contaminating lymphocytes can be treatedso as to interiorize drugs, proteins or antigens, by culture of saidmonocyte derived cells and contaminating lymphocytes for 2 to 24 h, inthe presence of drugs, proteins or antigens to interiorize thesecompounds in said monocyte derived cells.

In a particular embodiment of the invention, the process described abovecomprises, prior to the step of stimulation, a step of loading themonocyte derived cells with exogenous compounds such as drugs, proteins,growth factors of interest (e.g. by pinocytosis, phagocytosis ofparticular aggregates, diffusion), or with DNA coding for a protein ofinterest (i.e. with DNA plasmids, by sugar receptors mediated uptake forglycosylated polylysine-DNA or by lipid-DNA intake). The loaded monocytederived cells are then stimulated by physical means such as describedabove, and more particularly by electropulsation which causes thetransport of the exogenous compound loaded from the cytoplasm to thenuclei (where they can for example insert in DNA).

The process of the invention, in an advantageous embodiment, comprises,after the step of stimulation, the additional step of centrifugation ofthe stimulated monocyte derived cells at a temperature enabling cellpreservation, for instance at 4° C., and resuspension, for instance inisotonic medium containing autologous serum.

The process of the invention, according to another advantageousembodiment, comprises, after the step of stimulation, the additionalsteps of:

centrifugation of the stimulated monocyte derived cells at a temperatureenabling cell preservation, for instance at 4° C., and resuspension, forinstance in isotonic medium containing autologous serum, and

freezing at a temperature at least of −80° C. aliquots of the stimulatedmonocyte derived cells obtained at the preceding step, with the additionof a cryopreservative such as polyethyleneglycol, glycerol, DMSO(dimethylsulfoxide).

According to an advantageous embodiment, the process for the preparationof stimulated monocyte derived cells according to the invention,comprises the following steps:

loading the monocyte derived cells thus obtained with an exogenousnucleic acid through endocytosis targeting their mannose and/or Fcreceptors, or via pinocytosis of macromolecular nucleic acid aggregates,and

submission of the monocyte derived cells obtained at the preceding stepto physical stress such as electropulsation, for example about 1 toabout 10 pulses of about 5 msecs at about 0.3 to about 1 kV/cm, enablingintracellular transfer of the exogenous nucleic acid into the nucleusand integration into the DNA of the nucleus.

According to another advantageous embodiment of the invention, theprocess for the preparation of stimulated monocyte derived cellscomprises the following steps:

preparation of monocyte derived cells according to the following method:

1) recovery of blood derived mononuclear cells directly from bloodapheresis or from blood bag collection, followed if necessary bycentrifugation, to eliminate a substantial part of red blood cellsgranulocytes and platelets, and collection of peripheral bloodleukocytes;

2) washing peripheral blood leukocytes obtained at the preceding stepsfor instance by centrifugation (to remove 90% of platelets, red bloodcells and debris) to obtain mononuclear cells;

3) resuspension of the total mononuclear cells (monocytes+lymphocytes)obtained at the preceding step in culture medium (AIM-V, RPMI or IMDMtype) at 10⁶ to 2.10⁷ cells/ml, possibly completed by cytokines and/orautologous serum, and culture for 5 to 10 days at 37° C. under O₂/CO₂atmosphere in hydrophobic gas permeable bags, to obtain monocyte derivedcells and contaminating lymphocytes;

loading the monocyte derived cells thus obtained with an exogenousnucleic acid through endocytosis targeting their mannose and/or Fcreceptors, or via pinocytosis of macromolecular nucleic acid aggregates,and

submission of the monocyte derived cells obtained at the preceeding stepto physical stress such as electropulsation, enabling intracellulartransfer of the exogenous nucleic acid into the nucleus and integrationinto the DNA of the nucleus.

According to an advantageous embodiment of the invention, the processcomprises, after the step of electropulsation, the additional step ofcentrifugation of the stimulated monocyte derived cells at a temperatureenabling cell preservation, for instance at 4° C., and resuspension, forinstance in isotonic medium containing autologous serum.

According to another advantageous embodiment of the invention, theprocess comprises, after the step of electropulsation, the additionalsteps of:

centrifugation of the stimulated monocyte derived cells at a temperatureenabling cell preservation, for instance at 4° C., and resuspension, forinstance in isotonic medium containing autologous serum, and

freezing at a temperature at least of -80° C. aliquots of the stimulatedmonocyte derived cells obtained at the preceding step, with the additionof a cryopreservative such as polyethyleneglycol, glycerol, DMSO.

The invention also relates to stimulated monocyte derived cell such asobtained by the processes described above.

The invention also relates to a method for the ex-vivo stimulation ofmonocytes derived cells comprising physical stress. The stimulatedcells, as measured by biological effects generated, enhance the immuneresponse in vivo after reinjection to a patient.

The invention also relates to pharmaceutical compositions comprising, asactive substance, stimulated monocyte derived cells as described above,in association with a pharmaceutically acceptable vehicle.

Advantageous pharmaceutical compositions according to the invention, arein the form of sterile injectable preparations or of sterile topicalpreparations.

In the injectable preparation, the active substance is present in anamount such that it corresponds from about 10⁷ to about 10¹⁰ cells/kg ofbody weight, particularly from about 10⁸ to about 10⁹. In a topicalpreparation, the active substance is present in an amount of about 10⁵to about 10⁸ cells/cm² of body surface.

In particular embodiment, the monocyte derived cells are injectedrepeatedly at doses of 10⁷ to 5.10⁹ at intervals of 3 days to 6 months.

The injections can eventually be first local (subcutaneous,intramuscular, mucosal, in cavities or in tissues) and then systemic(intravenous or intralymphatic).

The invention also relates to pharmaceutical compositions as describedabove, in the form of a vaccine comprising, as active substance,stimulated monocyte derived cells as described above, having integratedin their nucleus an exogenous nucleic acid coding for a polypeptide orprotein which is immunogenic with respect to pathogens involved in thepathology to be treated.

The invention also relates to the use of stimulated monocyte derivedcells of the invention, for the preparation of a medicament for thetreatment of tissue repair

The invention also relates to a method for the treatment of tissuerepair comprising the use of stimulated monocyte derived cells asdescribed above.

The invention also relates to the use of stimulated monocyte derivedcells of the invention, for the preparation of a vaccine against tumorsor infectious agents, or of a medicament for treating polypeptide orprotein deficiency in a patient, said use comprising for instance thepreparation of sterile flasks of stimulated monocyte derived cellssuspension and their repeated local application on the injured site.

The invention also relates to a method for the vaccination againstturnouts or infectious agents comprising the use of stimulated monocytederived cells of the invention, with said stimulated monocyte derivedcells having integrated in their nucleus an exogenous nucleic acidcoding for a polypeptide or a protein which is immunogenic with respectto the above-mentioned tumor or infectious agent.

The invention also relates to a method for ex vivo gene therapycomprising the use of stimulated monocyte derived cells of theinvention, with said stimulated monocyte derived cells having integratedin their nucleus an exogenous nucleic acid coding for a polypeptide or aprotein which is deficient in a patient, said use comprising forinstance the preparation of a sterile injectable suspension ofstimulated monocyte derived cells and its repeated systemic and localinjection.

The invention also relates to a method for the stimulation of monocytederived cells comprising the preparation of stimulated monocyte derivedcells as described above, and injection in vivo to a patient tostimulate the immune system as evidenced by release of mediators andother biological effects.

The invention will be further illustrated in the following detaileddescription.

Ex vivo stressing of monocytes derived cells (MDC) by physical treatmentto induce a new desired stimulating biological activity.

Human blood derived mononuclear cells are grown ex vivo in culture bagsin defined medium. They are submitted to specific stimuli such aselectropulsation, heating at 40° C. to 50° C. or heat shock, microwaves.The intensity and length of these treatments determines thephysiological status achieved by the MDC (Monocytes Derived Cells).

Before physical treatment, the differentiated MDC have eventuallyphagocytosed specific compounds such as drugs, nucleic acids,polypeptides, chemokines or growth factors, and are loaded with thesecompounds to be processed and/or released when required. They havetherefore gained ex vivo new specific potential that can then beexploited therapeutically by local or systemic reinjection to thepatient from whom the original blood mononuclear cells were apherized.Thus the release of various factors artificially loaded or endogenouslyproduced by stressed MDC which are themselves in an activated status, iscontrolled.

Methods and culture conditions are disclosed describing the physicaltreatments used and the specific MDCs functionalities obtained. Thebeneficial effects of these cells used for the adoptive therapy ofspecific diseases are described.

Monocytes-Macrophages or Macrophages-Dendritic cells- grown ex vivo, aresubsequently stimulated by irradiation, electropulsation, or thermalstress for purpose of gaining new therapeutic stimulatingpotential—generally via controlled release of various factors eitherartificially loaded into or endogeneously produced by MDCs.

Monocytes derived cells can be obtained in large amounts (>10⁹ MDCs)after culture of mononuclear cells obtained from blood apheresis or fromblood “buffycoats” containing peripheral blood leucocytes in plastic orhydrophobic bags (for example ethylene vinylacetate or Teflon) and indefined culture media (see PCT patent application PCT/FR96/00121).

These MDCs are differentiated after one week of culture. They are thenexposed in vitro to physical stress.

In the present invention, the stress consists in the disturbance of thephysical environment of the cells (change of oxygen/CO₂ concentrationand pressure osmolality temperature change, electric stimulation,microwaves, ultrasonication . . . ) which results in temporarymodification of ion fluxes, activation of intracellular kinases,stimulation of stress proteins, flux of molecules (proteins, drugs,nucleic acids) from the cytoplasm to the nucleus.

The stimulated MDCs have therefore acquired new characteristics, asdescribed above.

TABLE 1 FACTORS RELEASED BY STRESSED STIMULATED MDC ENZYMES RADICALOXYGEN Lyzosymes Superoxide Neutral proteases Hydrogen peroxidePlasminogen activator Hydroxyl radical Collagenase Hypohalous acidsElastase BIOACTIVE LIPIDS Angiotensin-convertase Arachidonic acidmetabolites Acid hydrolases Prostaglandins E2, F2α ProteasesProstacyclin Lipases Thromboxane Ribonucleases Leukotrienes B4, C, D andE Phosphatases Hydroxy-eicosatetraeneoic acids Glycosidases (includingSRS-A) Sulphatases Platelet activating factors Arginase CYTOKINES,HORMONES COMPLEMENT COMPOUNDS Endogenous pyrogens C1, 4, 2, 3 and 5Interleukins 1α and β₁ Factors B and D and Properdin Tumors necrosisfactor α C1 inhibitor Interferons α and β₁ C3b inactivator and β-1HInterleukin 6 and 8 ENZYME INHIBITORS Chemotactic factors for(Antiproteases) Neutrophils α1-antiprotease T lymphocytes Plasmininhibitors Monocytes α2-macroglobulin Fibroblasts Plasminogen activatorinhibitors Hematopocetic Colony Stimulating PROTEINS BINDING FE Factorsfor AND LIPIDS Granulocyte-Macrophages Acidic isoferritins (GM-CSF)Transferrin Granulocytes (G-CSF) Transcobalamin II Macrophages (M-CSF)Fibronectin Erythropoeitin Laminin Growth factors Lipid transfer proteinFibroblast growth factor Thrombospondin “platelet-derived growth factor”Transforming growth factor α and β Endothelial cell growth factorHormones 1α, 25-Dihydroxyvitamin D3 Insulin-like activity Thymosin B4 βendorphin Adrenocorticotrophic hormone

EXAMPLES

Four examples of ongoing developments and applications are describedhereafter.

a) In a particular embodiment of the invention, MDCs are obtained afterone week of culture of cells with high phagocytic activity.

These MDCs are triggered by heating 30 minutes at 45° C. to express andrelease growth factors, heat shock and stress proteins, chemokines andmonokines. These cells added to cultures of human fibroblasts, of humanosteoblasts as well as to cultures of human chondrocytes stimulate theproliferation of these different cells.

The activated MDCs are frozen in aliquots at −80° C. (in 10% DMSO, 10%autologous serum or in polyethylene glycol 10% autologous serum) andthen used when needed. The concentration of polyethylene glycol can beincreased after fast unfreezing of the alilquot to obtain cells includedin a gel, which can be directly applied on wounds or tissues needingrepair. In vivo, these triggered MDCs sustain regeneration of skintissues presenting necrotic lesions.

The major portion of defrosted MDCs in 30% glycerol 20% autologous serumretains viability for at least 48 h in oxygenated conditions. When addedat a 1/1 ratio to fibroblasts, they increase their proliferation.Application of 10⁶ MDCs onto skin punch lesions induced to nude mice isused to assess the effect of these human MDCs on the quality ofcicatrisarion and of the detersion (histology).

The present invention describes in particular an effective method toinduce skin wound healing by local application of autologousmacropliages or MDCs prepared with the MAK Cell Processor(PCT/FR96/00121) and incorporated in an adequate pharmaceutical gelpreparation. Macrophages actively initiate phagocytosis of tissue debrisand contaminating bacteria. Simultaneously, they release locally fordays/weeks growth factors and monokines stimulating epidermal and dermaltissue regeneration and skin repair. These monokines are measured byELISA method.

In a particular embodiment of the invention the MDCs differentiated andrecovered with a cell processor are resuspended in glucose, polyethyleneglycol or sugar polymers (i.e.: Dextran derivatives, heparins or heparansulfate, mannose-6 phosphates). These sugar or polyethylene glycolpolymers allow cryopreservation of macrophage preparations aliquoted forsequential use, and after local application they stabilize bycomplexation of the growth factors secreted by macrophages and releasethem on demand. For cryopreservation of the macrophages, 4% autologousserum and 10% DMSO or polyethylene glycol can advantageously be used.

In another embodiment, macrophages are preloaded ax vivo with one orseveral growth factors such as PDGF, EGF, FGE . . . or a drug toincrease their wound healing potential.

In a particular embodiment, autologous macrophages are replaced fortherapy by an allogenic macrophage cell line such as Mono Mac 6 (ZieglerHeitbrock et al.—“Distinct patterns of differentiation induced in themonocytic cell line Mono Mac 6”, J. of Leucocyte Biol., Vol. 55, January1994), appropriately differentiated under good manufacture practiceconditions.

b) In another embodiment of the invention, the MDCs prepared accordingto the process described have interiorized tumor antigens of interest(by phagocytosis of tumor apoptotic bodies generated from tumor cellsand containing in particular mitochondria and DNA are cultured during 4h at 37° C. with the MDCs) and are then submitted to the physicalstress. Due to the induced presentation of the antigens at the same timeas accessory and costimulation molecules, specific T cells are activatedand do proliferate when cocultured with the MDCs. In vitro proliferationof lymphocytes is shown in the test of mixed lymphocyte proliferation.

Immunostimulation around the cells presenting the antigen of interest isdemonstated by the secretion of TH1 type cytokines (IL-12, IFNγ, IL-2)in the presence of stressed MDCs.

Vaccination against the antigen of interest is shown after the injectionof 1 million of these stressed antigen loaded MDCs by subcutaneous routein mice which causes potent immune response (presence of antitumorantibodies and of antitumor cytotoxic T lymphocytes).

c) In a third embodiment of the invention, MDCs are loaded with nucleicacids through endocytosis during 4 h at 37° C. of macromolecular nucleicacid mannosylated polylysine aggregates targetting their mannosereceptors. After washing and resuspension at 5 millions cells/ml inisotonic sucrose medium, these cells are then submitted to shortelectropulsation stimuli (5 pulses of 5 msecs at 0.5 kV/cm) allowingintracellular transfer of the nucleic acid from the cytoplasm into thecell nuclei and integration in DNA. These cells are then washed andinjected in animal models. The cell expression and local release forseveral weeks of the polypeptides coded by the nucleic acidsinteriorised before ex vivo physical treatment is demonstrated by ELISAand FACS analysis (fluorescence cell analysis).

Conditions for uptake of polylysine-cDNA are 0.1 μg/ml/10⁸ cells for 4 hat 37° C., followed by 5 square electric pulses of 5 millisec at 0.3 to0.5 kV.

The very effective transfection (≧20% efficiency and high intensity ofexpression) allows prolonged expression and release of the protein ofinterest in the extracellular medium.

This technique will prove particularly effective in the long lastingreplacement therapy for the treatment of generic deficiencies, forexample of Factor VIII in haemophiliacs with Factor VIII deficiency.

MDCs injected in an autologous way survive for several months in tissueswhere the release of factor of therapeutic interest can be measured.

d) Stimulation of monocyte derived cells by chemicals

Maturation and stimulation of MD-APC (monocyte derived-antigenpresenting cells).

MATERIAL AND METHODS Generation and Maturation of MD-APC

MD-APC are generated from total PBMC (Peripheral Blood MononuclearCells) obtained by apheresis and culture in AIM-V medium in the presenceof GM-CSF (500 UI/ml) and IL-13 (50 ng/ml). At day 7 of the culture,double stranded RNA (d.s.RNA )(30 μg/ml) is added to the culture and thecells are incubated for another 48 hours. Phenotypic characterization isperformed by flow cytometry on a FACScalibur cytofluometer using theCellQuest software (Becton Dickinson, San Jose, Calif.).

Allogenic Lymphocyte Proliferation

Serial dilutions (10³ to 10⁵) of mitomycine treated MD-APC are culturedin the presence of 10⁵ allogeneic responder T cells for 4 days. T cellproliferation is measured by BrdU incorporation.

RESULTS Phenotypic Maturation of MD-APC

Upon d.s.RNA treatment, CD40, CD80, CD86 expression increases on MD-APC.Moreover CD83, which is completely absent from untreated MD-APC isclearly expressed on more than 80% of cells cultured in the presence ofd.s.RNA. In contrast, CD14 which is expressed on 70% of the untreatedcells, is absent from cells treated with d.s.RNA.

Functional Maturation of MD-APC

Upon d.s.RNA treatment, 20 times less MD-APC are required to observe thesame level of T cell proliferation, as compared to untreated MD-APCindicating potent immunostimulatory properties.

Cytokine Secretion

IL-12 secretion by MD-APCs is increased at least 10 to 100 fold 48 hoursafter addition of d.s.RNA to the culture medium, which would favor TH1type immune responses.

What is claimed is:
 1. Isolated monocyte derived cells havingimmunostimulating properties and presenting the following properties:increased release, with respect to unstimulated monocyte derived cellswhich have not been exposed to chemical or physical stress, of IL12, andincreased presence, on their membranes, with respect to unstimulatedmonocyte derived cells, of the following molecules: MHC class I and MHCclass II molecules, accessory molecule CD40 and at least one of thefollowing activation markers CD80, CD83, and CD86.
 2. Isolated monocytederived cells having immunostimulating properties according to claim 1,loaded with exogenous compounds selected from the group consisting ofdrugs, proteins, growth factors and DNA coding for a protein. 3.Isolated monocyte derived cells having immunostimulating propertiesaccording to claim 1, wherein the activation markers are present in anamount of at least 1000 molecules/cells.
 4. Isolated monocyte derivedcells having immunostimulating properties according to claim 1, whereinthe polypeptides, proteins or compounds are released in an amount higherthan 1 pg/cell/hr and the activation markers are present in the range of10³ to 10⁵ molecules/cell.
 5. Process for conferring immunostimulatingproperties to macrophages, comprising the step of stimulating saidmacrophages by physical means selected from the group consisting ofthermal stress (heating at 40° C. to 50° C. for at least 30 minutes),pressure change (from 1 bar to 10 bars), microwaves, electric shock (1to 10 seconds at 250 mV), and electropulsation.
 6. Process for thepreparation of isolated monocyte derived cells having immunostimulatingproperties, comprising the steps: 1) recovering blood derivedmononuclear cells directly from blood apheresis or from blood bagcollection, followed by optional centrifugation, to eliminate asubstantial part of red blood cells, granulocytes and platelets, andcollection of peripheral blood leukocytes; 2) washing peripheral bloodleukocytes obtained at the preceding steps to obtain mononuclear cells;3) resuspending the cells (monocytes+lymphocytes) obtained at thepreceding step in culture medium (AIM-V, RPMI or IMDM type) at 10⁶ to2×10⁷ cells/ml, completed by cytokines and/or autologous serum, andculture for 5 to 10 days at 37° C. under O₂/CO₂ atmosphere inhydrophobic gas permeable bags, to obtain monocyte derived cells andcontaminating lymphocytes; stimulating said monocyte derived cells byphysical means selected from the group consisting of thermal stress(heating at 40° C. to 50° C. for at least 30 minutes), pressure change(from 1 bar to 10 bars), microwaves, electric shock (1 to 10s at 250mV), or electropulsation for a time sufficient to induce the stimulationof the cell or integration of exogenous nucleic acid into the DNA of themonocyte derived cell.
 7. Process for the preparation of isolatedmonocyte derived cells with immunostimulating properties, comprising thesteps: 1) recovering blood derived mononuclear cells directly from bloodapheresis or from blood bag collection, followed by optionalcentrifugation, to eliminate a substantial part of red blood cells,granulocytes and platelets, and collection of peripheral bloodleukocytes; 2) washing peripheral blood leukocytes obtained at thepreceding steps to obtain mononuclear cells; 3) resuspending the cells(monocytes+lymphocytes) obtained at the preceding step in culture medium(RPMI or IMDM type) at 10⁶ to 2×10⁷ cells/ml, possibly completed bycytokines and/or autologous serum, and culture for 5 to 10 days at 37°C. under O₂/CO₂ atmosphere in hydrophobic gas permeable bags, to obtainmonocyte derived cells and contaminating lymphocytes; stimulating saidmonocyte derived cells by addition of chemicals, which induce IFNproduction said chemicals being selected from the group consisting ofdouble stranded RNA, bacterial or mycobacterial extracts and bacterialtype DNA.
 8. Process for the preparation of isolated monocyte derivedcells with immunostimulating properties according to claim 6,comprising, before the step of stimulating, the step of culturing ofsaid monocyte derived cells and contaminating lymphocytes for 2 to 24hours, in the presence of drugs, proteins or antigens to interiorizethese compounds in said monocyte derived cells.
 9. Process for thepreparation of isolated monocyte derived cells with immunostimulatingproperties according to claim 6, comprising the additional step ofcentrifuging the isolated monocyte derived cells with immunostimulatingproperties at a temperature of 4° C., said temperature enabling cellpreservation, and resuspension in isotonic medium containing autologousserum.
 10. Process for the preparation of isolated monocyte derivedcells with immunostimulating properties according to claim 6, comprisingthe additional steps of: centrifuging the isolated monocyte derivedcells with immunostimulating properties at a temperature of 4° C., saidtemperature enabling cell preservation, and resuspending the isolatedmonocyte derived cells with immunostimulating properties in isotonicmedium containing autologous serum, and freezing at a temperature of atleast −80° C. aliquots of the isolated monocyte derived cells withimmunostimulating properties obtained at the preceding step, adding acryopreservative selected from the group consisting of polyethyleneglycol, glycerol, and DMSO (dimethylsulfoxide).
 11. Process for thepreparation of isolated monocyte derived cells with immunostimulatingproperties comprising the steps: loading the monocyte derived cellsobtained by claim 9 with an exogenous nucleic acid through endocytosistargeting their mannose and/or Fc receptors, or via pinocytosis ofmacromolecular nucleic acid aggregates, and submitting the monocytederived cells obtained at the preceding step to physical stressincluding electropulsation from 1 to 10 pulses of 5 msecs at 0.3 to 1kV/cm, enabling intracellular transfer of the exogenous nucleic acidinto the nucleus and integration into the DNA of the nucleus. 12.Process for the preparation of isolated monocyte derived cells withimmunostimulating properties comprising the steps: 1) recovering bloodderived mononuclear cells directly from blood apheresis or from bloodbag collection, followed by optional centrifugation, to eliminate asubstantial part of red blood cells, granulocytes and platelets, andcollection of peripheral blood leukocytes; 2) washing peripheral bloodleukocytes obtained at the preceding steps to obtain mononuclear cells;3) resuspending the cells (monocytes+lymphocytes) obtained at thepreceding step in culture medium (AIM-V, RPMI or IMDM type) at 10⁶ to2×10⁷ cells/ml, possibly completed by cytokines and/or autologous serum,and culture for 5 to 10 days at 37° C. under O₂/CO₂ atmosphere inhydrophobic gas permeable bags, to obtain monocyte derived cells andcontaminating lymphocytes; loading the monocyte derived cells thusobtained with an exogenous nucleic acid through endocytosis targetingtheir mannose and/or Fc receptors, or via pinocytosis of macromolecularnucleic acid aggregates, and submitting the monocyte derived cellsobtained at the preceding step to electropulsation, enablingintracellular transfer of the exogenous nucleic acid into the nucleusand integration into the DNA of the nucleus.
 13. Process for thepreparation of isolated monocyte derived cells with immunostimulatingproperties according to claim 11, comprising, before the step ofloading, the step of culturing said monocyte derived cells andcontaminating lymphocytes for 2 to 24 hours, in the presence of drugs,proteins or antigens to interiorize these compounds in said monocytederived cells.
 14. Process for the preparation of isolated monocytederived cells with immunostimulating properties according to claim 12,comprising the additional step of centrifuging the monocyte derivedcells with immunostimulating properties at a temperature of 4° C., saidtemperature enabling cell preservation, and resuspending the cells inisotonic medium containing autologous serum.
 15. Process for thepreparation of isolated monocyte derived cells with immunostimulatingproperties according to claim 12, comprising the additional steps of:centrifuging the monocyte derived cells with immunostimulatingproperties at a temperature of 4° C., said temperature enabling cellpreservation and resuspending the monocyte derived cells in isotonicmedium containing autologous serum, and freezing at a temperature of atleast −80° C. aliquots of the monocyte derived cells withimmunostimulating properties obtained at the preceding step, with theaddition of a cryopreservative selected from the group consisting ofpolyethylene glycol, glycerol, and DMSO (dimethyl-sulfoxide). 16.Pharmaceutical composition comprising, as active substance, isolatedmonocyte derived cells with immunostimulating properties according toclaim 1, in association with a pharmaceutically acceptable vehicle. 17.Pharmaceutical composition according to claim 16, in the form of sterileinjectable preparations or of sterile topical preparations. 18.Pharmaceutical composition in the form of a vaccine comprising, asactive substance, isolated monocyte derived cells with immunostimulatingproperties according to claim 1, having integrated in their nucleus, anexogenous nucleic acid coding for a polypeptide or protein which isimmunogenic.
 19. Medicament comprising isolated monocyte derived cellswith immunostimulating properties according to claim 1, in the form of avaccine against tumors or infectious agents, and for treatingpolypeptide or protein deficiency in a patient.